Enzyme immunoassay: overview (2023)

Enzymgebundene Immunoassays

Commercially available EIAs are commonly used to detect toxins A and BIt is difficultin stool samples.^23,26,124The toxin is detected by its interaction with a monoclonal antibody or polyclonal antiserum that specifically recognizes the toxin's epitopes. EIAs are easier to perform than cytotoxicity testing, are relatively inexpensive, and are quick, with results in 1 to 6 hours. Although they have a high specificity (83% to 98%), their main disadvantage is that they are less sensitive (75% to 95%) than the cytotoxicity test.^124,125; As such, the toxin EIA is now rarely used as a standalone test, but is usually combined with a more sensitive assay (such as GDH or NAAT). Also, some EIA kits only detect toxin A, in this case diarrhea caused by a toxin A⁻/B⁺tribe ofIt is difficultwill have a false negative test result.^76.126For this reason, assays that can detect both toxin A and toxin B are preferred.

hepatitis-e-virus

Enzymimmunoassaysfor HEV IgM and IgG are commercially available in Europe and Asia in microplate and automated multi-parameter instrument formats (Tabla 2). Various immunochromatographic assays are also available in different countries. The antigens used are usually recombinant ORF2 capsid protein and/or ORF3 protein from the HEV genotype 1 strain. The diagnostic performance of these immunoassays varies widely and must be carefully evaluated.

The presence of HEV IgM in serum is a key marker of acute infection. Using a validated PCR assay as a reference, studies have shown that the sensitivity of IgM immunoassays is >97% for immunocompetent patients and 80%-85% for immunocompromised patients; its specificity is >99.5%. Long-term follow-up of patients with nucleic acid-proven acute hepatitis E showed that HEV IgM persisted for 6 to 12 months.

The presence of HEV IgG alone is a marker of past infection. It is difficult to compare seroprevalence rates for different populations determined by different laboratory methods because the analytical sensitivity of IgG immunoassays varies widely. Detection limits of commercial HEV IgG assays range from 0.25 WHO units/mL to 2.5 WHO units/mL when measured with an international standard (WHO reference reagent introduced in 2002; National Institute for Standards and Biological control, code 95/584). As highlighted in a meta-analysis of HEV seroprevalence studies in Europe, the most sensitive immunoassay provided the highest estimates of HEV IgG seroprevalence. Determining the HEV IgG concentration could be helpful in estimating the risk of reinfection after natural infection or vaccination.

Enzymgebundene Immunoassays

Enzymimmunoassaysfor coccidioidal antibodies are commercially available (Meridian Bioscience, Inc., Cincinnati, OH; IMMY, Norman OK; Mira Vista Diagnostics, Indianapolis, IN). The test kits enable the specific detection of IgM or IgG antibodies; however, these results are not interchangeable with results from immunodiffusion or complement fixation tests. Positive results with this commercial kit are very sensitive to coccidia infection. False positive results are occasionally observed, particularly with the IgM enzyme immunoassay.^179–181Currently, enzyme immunoassay results usually need to be confirmed with precipitin immunodiffusion tubes, complement fixation immunodiffusion, or complement fixation test results. If more established tests fail to confirm the enzyme immunoassay, coccidioid infection may indeed be present, but the diagnosis is less certain.^182–184

Enzyme-Linked Immunosorbent Assays (ELISA)

ELISA methods have been developed for a variety of applications in veterinary medicine. On one level, veterinarians themselves have commercialized kits for the rapid diagnosis of several important viral diseases. At another level, ELISA procedures have been automated with the introduction of automated dispensing, washing, and spectrophotometric reading and recording instruments, making it possible to process hundreds of samples in a day, for example in pig pseudorabies antibody testing.

Diagnose

Diagnosis of CDI is based on the presence of clinical symptoms (generally defined as ≥ 3 watery, loose, or unformed stools in a 24-hour period or less) along with a diagnostic test (usually a stool sample) that demonstrates the presence of either theIt is difficultOrganism or its toxin genes, detection ofIt is difficultToxin mit aEnzymimmunoassayor cellular cytotoxin assay. Diagnosis can also be made in patients with symptoms of diarrhea using lower GI endoscopy to visualize pseudomembranes in the colon, but this method is used sparingly and is much more expensive and less sensitive than a variety of stool diagnostics.Tabla 243.3). The presence of appropriate symptoms before the stool test is crucial becauseIt is difficultit can be worn asymptomatically, especially by hospitalized patients.¹⁸¹Since the pathogen and its toxins can be easily detected with most available tests, the detection ofIt is difficultwithout symptoms does not meet criteria for a CDI diagnosis.

There is considerable controversy surrounding the most appropriate stool test for diagnosing CDI. The first test used and one of the gold standards was the cellular cytotoxicity test, which was developed concurrently with the discovery ofIt is difficultand its toxins as a cause of CDI.¹⁸²The test is performed by taking dilutions of stool supernatants, placing them in cell culture lines (a variety of cell types can be used) and incubating for 24 to 48 hours, then observing microscopically for cell rounding, an indication of the presence of toxin B (and occasionally toxin A). Confirmation that the cell rounding is due to thisIt is difficultToxin is produced by neutralizing the assay withIt is difficultÖClostridium sordelliiAntitoxin (the latter cross-reacting withIt is difficulttoxins).

Shortly after the cell cytotoxicity assay was developed, a selective medium containing cycloserine and cefoxitin was developed for the cultivation ofIt is difficultof faeces, paves the way for the second gold standard test, culture of faeces for toxigeneIt is difficult.¹⁸³The first enzyme immunoassay (EIA) for toxin A was described in 1983, followed by the development of a monoclonal antibody to toxin A that set the standard for CDI testing for decades to come. Although not as sensitive as the two gold standard tests, the cell cytotoxicity test and the toxigenic culture (culture ofIt is difficultof faeces and confirmation of toxin production in vitro), EIA required less work and provided faster results.^184.185

After decades of use, the EIA test for toxin A became obsolete after failing to detect the increasing clinical outbreaks of CDI in multiple hospitals caused byIt is difficultA−/B+ organisms that do not produce toxin A but cause CDI.¹⁸⁶The toxin A EIA test was later replaced by a variety of toxin A/B EIA tests that use a monoclonal antibody to detect toxin A and polyclonal antibodies to detect toxin B. These assays are relatively insensitive compared to the cytotoxicity assay Toxigenic Cell Culture Test.It is difficult(verTabla 243.3).^187.188Not only are EIA assays less sensitive than cell cytotoxicity assays, but cell cytotoxicity assays are also less sensitive than toxigenic cultures.^189.190

ENZYME IMMUNORBENT ASSAY (ELISA OR EIA)

sleepieret al.(1978)develops a responsive, fast and easyEnzymimmunoassayto detect both the hepatitis A virus and its antibody. Hepatitis A antigen binds to the walls of polyvinyl chloride or polystyrene microtiter plates coated with hepatitis A antibodiesThe antibody conjugated to horseradish peroxidase is then allowed to react with the bound antigen. An enzyme substrate is used and the color reaction can be read with the naked eye or with a spectrophotometer. The sensitivity of this technique is comparable to that of the radioimmunoassay.Matthiaset al.(1978)also found that the sensitivity of the enzyme immunoassay was comparable to that of solid-phase radioimmunoassay and electron immunomicroscopy for the detection of hepatitis A virus in stool and hepatitis A antibody in serum. Since human faeces often react non-specifically in serological tests for hepatitis A virus, blocking the reaction with hyperimmune antibodies against hepatitis A produced in a chimpanzee was used to confirm the specificity of the test.Lokarniniet al.(1978)reported that the enzyme immunoassay is an accurate and rapid means of detecting hepatitis A virus in stool. There was no appreciable loss of sensitivity by shortening the incubation time to 4 h (2 h for antigen binding and 2 h for blocking the reaction to determine specificity), allowing samples to be tested the day after collection. It was also found that this technique allows rapid detection of large numbers of samples with ease, and if stool samples are available within a week of the onset of dark urine, virus detection could have rapid diagnostic value. In addition, since the test is relatively inexpensive, the reagents have a long shelf life, no complex technical equipment is required, and the results can be read with the naked eye, the test is suitable for large-scale epidemiological studies. In this context,Crovariet al.(1978)noted that radioimmunoassay has improved over the years, while enzyme immunoassay is a relatively recent innovation with considerable potential. In addition, some countries, including Italy, have enacted laws restricting the use, handling and disposal of radioactive material, making the enzyme immunoassay an attractive alternative test system.

Reading procedure for polymerase chain reactions

Amplification products can be detected by hybridization with a probe mix in a DNA enzyme immunoassay (DEIA/EIA) in a microtiter plate. In addition, reverse hybridization (RH) can be performed using separate genotype-specific probes immobilized on supports such as nylon membranes, nitrocellulose strips, microbeads, or DNA chips. Readout is typically achieved via labeled or tagged probes or targets and enzyme or fluorochrome detection, and is typically performed in a real-time format as opposed to conventional endpoint PCR, particularly for high throughput assays. PCR product readers can be designed to detect multiple HPV types simultaneously (detection), individually (complete genotyping), or as a combination of detection and genotyping (partial genotyping).

14.1 Origins and introduction to mass spectrometry in mycology

New diagnostic techniques in the recent history of modern microbiology, e.g. B. the techniques ofEnzymimmunoassayin the early 1970s or techniques of molecular microbiology (polymerase chain reaction; PCR) have become a real advantage in the daily life of clinical microbiology laboratories in the last decades. These techniques have greatly improved their diagnostic capacity, speed of results, and sensitivity and specificity. In addition, these could be integrated into automated systems, and the current progressive increase in routine processes[1].

Like the techniques mentioned above, the introduction of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) was undoubtedly the most important technological advance in clinical microbiology during the last decade. In just a few years, it has become a widespread technology for everyday clinical use, available in the microbiology services of various hospitals.

In 2009, a key article was published to bring the possibilities of MALDI-TOF-MS to the attention of specialists involved in diagnosing infectious diseases.[2]. More than 1600 isolates were analyzed in these studies, including gram-positive and gram-negative, aerobic and anaerobic bacteria, with 95.4% correct identification.

On the other hand, severe fungal infections, which often affect particularly sensitive patients, such as B. immunosuppressed and critical patients, an early establishment of an adequate antifungal therapy, so that a quick diagnosis is essential for a good prognosis.

The identification of isolated organisms (like yeasts) has traditionally been performed by enzymatic, biochemical and morphological studies, or in the case of filamentous fungi mainly by morphological studies.[3]. Generally, these methods require subculture or biochemical testing, which delays the microbiological outcome for more than 24 hours after isolation of the pathogen. The speed, safety and simplicity of the microbial identification procedures offered by MALDI-TOF MS make it the ideal tool in the clinical laboratory context.[4].

In recent years, numerous studies have demonstrated the benefits of MALDI-TOF MS for fungal identification.[5,6]. In fact, its limitations are more related to the availability of a complete database than to the method's ability to obtain reliable profiles of almost any organism.[7].

In this chapter we will discuss key aspects of MS in identifying the main clinical types, as well as other applications of great interest in the field of mycology and their possible use in epidemiological studies or studies related to antifungal susceptibility.

diagnosis and prevention

Viral RNA can usually be detected in the blood of HCPS and HFRS patients by polymerase chain reaction using samples collected during the first week of illness, which is useful as it also identifies the genotype of the infecting virus.

Vaccines against hantavirus infection have been used in China and Korea for years, but not in Europe or America. A pan-hantavirus vaccine candidate has been reported to offer protection in animal models but is not yet in clinical trials. No specific antiviral therapy is used in Europe, but ribavirin and interferon-α have been used in studies in China. A single dose of icatibant, a bradykinin receptor antagonist, has been used successfully to treat patients with severe HFRS caused by Puumala. A major problem is that by the time the patient is admitted to the hospital, the rate of viral replication is already decreasing, so reducing viral replication with antiviral drugs is no longer necessary for the patient. Therefore, prevention of hantavirus infection remains dependent on reducing exposure to feces of infected rodents.

Solid Phase Microplate Immunoassays

Competitive Immunoassays: In competitive immunoassays (Figure 2 (B)) the labeled monoclonal or polyclonal antibody generates the signal by binding to its antigen. If a sample contains antibodies against the same epitopes, these compete with the labeled antibody and prevent it from binding. This leads to a lower signal and the degree of this inhibition is proportional to the amount of specific antibodies in the sample. Competitive immunoassays are highly specific in that nonspecific or cross-reactive antibodies that bind to other epitopes will not be detected unless they bind tightly enough to cause steric hindrance. As long as suitable monoclonal antibodies exist, competitive immunoassays can measure antibodies against a single epitope (e.g., a neutralizing epitope) without having to separate that epitope from the rest of the antigen (which may not be feasible). As such, competitive assays cannot distinguish between immunoglobulin glasses, but IgG can be removed or precipitated from the sample to reveal possible underlying IgM reactivity.

Capture immunoassays: Instead of antigens, the solid phase of capture immunoassays (Figure 2(C)) consists of anti-IgM or anti-IgG capture antibodies (or IgG-binding proteins such as protein A or G) that bind to and “capture” the entire IgM or IgG populations in the sample. The labeled antigen is then added and only binds to antibodies specific for it if they are present in the captured immunoglobulin population. This makes the method very specific, although cross-reactive antibodies can still lead to false alarms. With IgG, only a small fraction of the total population of captured antibodies is specific for a given antigen, which can cause sensitivity problems if the signal is not sufficiently amplified. This is less of a problem with IgM, as it occurs primarily in acute infections and usually does not persist thereafter. Capture immunoassays are also unaffected by rheumatoid factor, making them particularly useful for IgM diagnostics.